The Role of the 3’ UTR of Dulcamara mottle virus

RNA in Translation

Alma Laney

Dr. Yannis Tzanetakis

Dr. Theo Dreher

mRNA Translation

mRNA structure5’ cap 3’ UTR

60s

40s

An

An

Enzyme

mRNA translation scheme

Positive Strand RNA Viruses

VPg

mRNA, Flexiviruses,Togaviruses

TLS Tymo-, Tobamoviruses

Flaviviruses, Closteroviruses

AnPicornaviruses,

Potyviruses

Barley yellowdwarf virus

5´cap: m7G(5´)ppp(5´)-N

An

RNA virus translation• How do the untranslated regions (UTR) of RNA

viruses enhance translation?

TYMV translation

• The TLS of TYMV mimics a tRNA.

TYMV and DuMV genomes

DuMV

MP (62 kD) CP (20 kD)

RP (196 kD)* MTR PRO HEL POL

129 nt5´-UTR

248 nt3´-UTR

RNAi suppressor

MP

* R HE

Pr teolyticMaturat oCleava e

OL

(69 kD)

p141 p66

CP (20 kD)

RP (206 kD)TLS (Val)MTR P O L

oi n

g

P

RNAi suppressor

TYMV

87 nt5´-UTR

109 nt3´-UTR

TYMV and DuMV 3’ termini

CUC

GA

UC

C

AA

C C

GAGCCUCG

CGUCGCAG

U

GCGACGCU

UUAAA

UUC

U

5´ Dulcamara mottle virus3´- pseudoknotA

AU

G-CC-GG-CA-UG-CGU-AC-GU-AG-CU-AC-G

AC

UA

CCCC A C

A

UU G

AA

C U

C G U

CCCGGGGC

CCCGGG

CUCU

UCGGAAGCCU

UCA

T

D

A/C

ACC(A)

5´

TYMV TLS

Quic

kTim

e™

and

aTIF

F (

Un

com

pre

ssed

) d

ecom

pre

ssor

are

ne

ede

d t

o s

ee t

his

pic

ture

.

The 3’ UTR of DuMV

DuMV 248 nt

44 nt 59 nt 145 nt

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAACAAAACAAAACAAA

CUC

GA

UC

C

AA

C C

GAGCCUCG

CGUCGCAG

U

GCGACGCU

UUAAA

UUC

U

5´ Dulcamara mottle virus

3´- pseudoknot

The Question

• Does the 3’ UTR of DuMV enhance translation?

44 nt 59 nt 145 nt

The Hypothesis

• The combination of the 59 nt and 145 nt sequences are responsible for translational enhancement.

59 nt 145 nt

(Pseudoknot)( Poly A Tail)( Poly A Tail)

Methodology

• Six plasmid constructs were created with the luciferase gene.

• The plasmids will be tested to see luciferase expression.

luciferase 3’ UTR luciferase reaction Light generated

Luciferin + ATP

Plasmid constructs

LuciferaseLuciferase Spacer

LuciferaseLuciferase Spacer A Track

LuciferaseLuciferase A Track

LuciferaseLuciferase Pseudoknot

LuciferaseLuciferase A Track Pseudoknot

LuciferaseLuciferase A TrackSpacer Pseudoknot

2. Ligation of UTR fragment to pLUC

1. PCR of UTR section

Creating the plasmid constructs

3. Transformation 4. Restriction Enzyme Digest

Creating the plasmid constructs cont.

5. Gel Electrophoresis 6. DNA sequencing

Creating the plasmid constructs cont.

2. In vitro transcription1. Linearize plasmid

3. RNA transfection

5. Cell lysis

6. Luciferase reaction

LUC

Cowpea protoplasts

*

4. Incubation under light

+/- DuMV UTR fragments

Luciferase assay to test for translational enhancement

Results

XXLuc + A-tail

XXLuc + Spacer + A Tail

XXLuc + Spacer

AssayedSequencedCloned

Luc + complete

Luc + A-tail + Pseudoknot

Luc + Pseudoknot

X

x

X

X

X

0

2 104

4 104

6 104

8 104

1 105

1.2 105

1.4 105

RLU

0 1 2 3 4 76 85 9

Time (hrs)

Example of Luciferase Assay

Luc

Luc

Luc

Luc

LucSpacer A Track Pseudoknot

Future Work

• The next step is to create several genome chimeras to test infectivity in plants.

DuMV Complete Genome TYMV TLS

TYMV Complete Genome DuMV 3’ UTR

Acknowledgements

• Thanks to the Howard Hughes Medical Institute.

• Dr. Yannis Tzanetakis

• The Theo Dreher Lab